DNA polymerases synthesize DNA molecules in the 5′ to 3′ direction from deoxynucleoside triphosphates (nucleotides) using a complementary template DNA strand and a primer by successively adding nucleotides to the free 3′-hydroxyl group of the growing strand. The template strand determines the order of addition of nucleotides via Watson-Crick base pairing. In cells, DNA polymerases are involved in DNA repair synthesis and replication (Kornberg, 1974, In DNA Synthesis. W. H. Freeman, San Francisco).
Archaeal DNA polymerases have a 3′ to 5′ exonuclease activity and a DNA synthesis activity. Many molecular cloning techniques and protocols involve the synthesis of DNA in in vitro reactions catalyzed by DNA polymerases. Sometimes, mutant forms of DNA polymerases are desired for particular uses. For example, DNA polymerases are used in DNA labelling and DNA sequencing reactions, using either 35S-, 32P- or 33P-labelled nucleotides. Most of these enzymes require a template and primer, and synthesize a product whose sequence is complementary to that of the template. The 5′ to 3′ exonuclease activity of An Archaeal DNA polymerases is often troublesome in these reactions because it degrades the 5′ terminus of primers that are bound to the DNA templates and removes 5′ phosphates from the termini of DNA fragments that are to be used as substrates for ligation. The use of DNA polymerase for these labelling and sequencing reactions thus may depend upon the removal of the 5′ to 3′ exonuclease activity.
DNA processivity is performed by heat denaturation of a DNA template containing the target sequence, annealing of a primer to the DNA strand and extension of the annealed primer with a DNA polymerase. The concept of net DNA processivity is the ratio of DNA synthesis activity versus 3′-5′ exonuclease activity (for reviews, see, e.g., Kelman et al., 1998 Processivity of DNA polymerases: two mechanisms, one goal. Structure 6(2):121-5; Wyman and Botchan, 1995, DNA replication. A familiar ring to DNA polymerase processivity. Curr Biol. 5(4):334-7; and Von Hippel et al., 1994, On the processivity of polymerases. Ann N Y Acad Sci. 726:118-31). DNA synthesis activity acts to polymerize nucleotides while 3′-5′ exonuclease has an editing or proof-reading function to enhance the fidelity of the synthesis. Thus highly efficient DNA synthesis is generally achieved at the expense of high fidelity and vice versa. The 3′-to-5′ exonuclease activity of many DNA polymerases may, therefore, be disadvantageous in situations where one is trying to achieve net synthesis of DNA and/or where fidelity is not of primary concern.
Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an G-C to A-T mutation (Fogg et al., 2002, Nat Struct Biol. 9(12):922-7). Uracil detection is thought to represent the first step in a pathway to repair DNA cytosine deamination (dCMP→dUMP) in archaea (Greagg et al, 1999, PNAS USA, 96:9405). Stalling of DNA synthesis opposite uracil has significant implications for high-fidelity PCR amplification with Archaeal DNA polymerases. Techniques requiring dUTP (e.g., dUTP/UDG decontamination methods, Longo et al. 1990, Gene, 93:125) or uracil-containing oligonucleotides can not be performed with proofreading DNA polymerases (Slupphaug et al. 1993, Anal. Biochem., 211:164; Sakaguchi et al. 1996, Biotechniques, 21:368). But more importantly, uracil stalling has been shown to compromise the performance of Archaeal DNA polymerases under standard PCR conditions (Hogrefe et al. 2002, PNAS USA, 99:596).
During PCR amplification, a small amount of dCTP undergoes deamination to dUTP (% dUTP varies with cycling time), and is subsequently incorporated by Archaeal DNA polymerases. Once incorporated, uracil-containing DNA inhibits Archaeal DNA polymerases, limiting their efficiency. We found that adding a thermostable dUTPase (dUTP→dUMP+PPi) to amplification reactions carried out with Pfu, KOD, Vent, and Deep Vent DNA polymerases significantly increases PCR product yields by preventing dUTP incorporation (Hogrefe et al. 2002, Supra). Moreover, the target-length capability of Pfu DNA polymerase is dramatically improved in the presence of dUTPase (from <2 kb to 14 kb), indicating that uracil poisoning severely limits long-range PCR due to the use of prolonged extension times (1-2 min per kb @72° C.) that promote dUTP formation.
In addition to dUTP incorporation, uracil may also arise as a result of cytosine deamination in template DNA. The extent to which cytosine deamination occurs during temperature cycling has not been determined; however, any uracil generated would presumably impair the PCR performance of Archaeal DNA polymerases. Uracil arising from cytosine deamination in template DNA is unaffected by adding dUTPase, which only prevents incorporation of dUTP (created by dCTP deamination). Adding enzymes such as uracil DNA glycosylase (UGD), which excise uracil from the sugar backbone of DNA, or mismatch-specific UDGs (MUG), which additionally excise G:T mismatches, is one way to eliminate template uracil that impedes polymerization.
Alternatively, the problem of uracil stalling may be overcome by introducing mutations or deletions in Archaeal DNA polymerases that reduce, or ideally, eliminate uracil detection, and therefore, allow synthesis to continue opposite incorporated uracil (non-mutagenic uracil) and deaminated cytosine (pro-mutagenic uracil). Such mutants would be expected to produce higher product yields and amplify longer targets compared to wild type Archaeal DNA polymerases. Moreover, mutants that lack uracil detection should be compatible with dUTP/UNG decontamination methods employed in real-time Q-PCR.
It is sometimes desired for a DNA polymerase or a reverse transcriptase to have a high processivity. Processivity is a measurement of the ability of a DNA polymerase to incorporate one or more deoxynucleotides into a primer template molecule without the DNA polymerase dissociating from that molecule. DNA polymerases having low processivity, such as the Klenow fragment of DNA polymerase I of E. coli, will dissociate after about 5-40 nucleotides are incorporated on average. Other polymerases, such as T7 DNA polymerase in the presence of thioredoxin, are able to incorporate many thousands of nucleotides prior to dissociating. In the absence of thioredoxin such a T7 DNA polymerase has a much lower processivity. Processivity factors have been identified to increase the processivity of a DNA polymerase (e.g., see Carson D R, Christman M F. 2001, Proc Natl Acad Sci U S A. 98(15):8270-5).
U.S. Pat. No. 5,972,603 teaches a chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with the DNA polymerase domain, where the processivity factor binding domain binds thioredoxin.
U.S. patent application with Ser. No. 2002/0119467 describes a method for increasing the processivity of reverse transcriptase (RT) E. coli DNA polymerase and T7 DNA polymerase using a polynucleotide binding protein such as Ncp7, recA, SSB and T4gp32.
There is therefore a need for thermostable DNA polymerases that can amplify DNA in the presence of dUTP without compromising proofreading or polymerization activity and efficiency. There is also a need for thermostable DNA polymerases that can amplify DNA efficiently without the proof checking function of 3′-5′ exonuclease activity so that the thermostable DNA polymerase exhibits increased processivity.